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Two taxonomic and nomenclatural issues in Lithospermum are addressed. The new combination L. thurberi based on Onosmodium thurberi is made, and L. macromeria (Macromeria viridiflora) is included as a heterotypic synonym. Lithospermum bejariense and Onosmodium bejariense are treated here as two distinct taxonomic entities. [ABSTRACT FROM AUTHOR]

Objective: To investigate the optimal dose of mifepristone and lithospermum combination regimen on medical abortion in early pregnancy rats without increasing side effects. Study Design: Sixty sexually mature female Sprague Dawley (SD) rats with early pregnancy were randomly allocated into 10 groups, including a control group (treated with 0.5% CMC‐Na) and nine experiments (treated with 1 mg/kg mifepristone, and 90, 180, 270, and 540 mg/kg lithospermum, and 90/180/270/540 mg/kg lithospermum +1 mg/kg mifepristone, respectively). The hormone levels, factors associated with endometrial bleeding, oxidative stress, and apoptotic proteins in the endometrium, were then investigated. Results: The results demonstrated that 540 mg/kg lithospermum plus 1 mg/kg mifepristone treatment significantly improved the abortion rate when compared with the control group. Compared with the 1 mg/kg mifepristone, 540 mg/kg lithospermum plus 1 mg/kg mifepristone treatment did not induce significant increase in factors associated with abnormal endometrial bleeding, such as matrix metalloproteinase‐9 (MMP9). However, mifepristone and lithospermum combination regimen promoted the expression level of malondialdehyde (MDA), activated caspase 3, caspase 9 and Bax, meanwhile, reduced the expression of superoxide dismutase (SOD) and Bcl‐2. Conclusion: These findings provided strong evidence that mifepristone and lithospermum combination regimen can obtain satisfactory abortion effect without increasing the expression level of bleeding‐related factors. [ABSTRACT FROM AUTHOR]

Shikonin and its derivatives are the main components of traditional Chinese medicine, Zicao. The pharmacological potential of shikonin and its derivatives have been extensively studied. Yet, less is known about the microbial assemblages associated with shikonin producing Borage plants. We studied microbial profiles of two Borage species, Echium plantagineum (EP) and Lithospermum erythrorhizon (LE), to identify the dynamics of microbial colonization pattern within three rhizo-compatments and two distinct soil types. Results of α and β-diversity via PacBio sequencing revealed significantly higher microbial richness and diversity in the natural soil along with a decreasing microbial gradient across rhizosphere to endosphere. Our results displayed genotype and soil type–dependent fine-tuning of microbial profiles. The host plant was found to exert effects on the physical and chemical properties of soil, resulting in reproducibly different micro-biota. Analysis of differentially abundant microbial OTUs displayed Planctomycetes and Bacteroidetes to be specifically enriched in EP and LE rhizosphere while endosphere was mostly prevailed by Cyanobacteria. Network analysis to unfold co-existing microbial species displayed different types of positive and negative interactions within different communities. The data provided here will help to identify microbes associated with different rhizo-compartments of potential host plants. In the future, this might be helpful for manipulating the keystone microbes for ecosystem functioning. [ABSTRACT FROM AUTHOR]

Objective: To explore the mechanism of Radix Astragali-Lithospermum Erythrorhizon on the treatment of diabetic ulcer through the method of network pharmacology. Methods: This study included 32 compounds and 81 key targets. 100 GO functional items and 116 KEGG signal pathways were obtained by enrichment analysis. Quercetin, kaempferol, isorhamnetin, mononetin, sitosterol, ivy sapogenin and other components of astragalus-purple herb play a key role in the targets of interleukin-6, cystatin 3, vascular endothelial growth factor, epidermal growth factor receptor and mitogen-activated protein kinase 8 in diabetic ulcer, and are mainly concentrated in AGE-RAGE, TNF and other signal pathways. Results: There were 32 compounds and 81 key targets. 100 GO functional items and 116 KEGG signal pathways were obtained by enrichment analysis. Quercetin, kaempferol, isorhamnetin, mononetin, sitosterol, ivy sapogenin and other components of astragalus-purple herb play a key role in the targets of interleukin-6, cystatin 3, vascular endothelial growth factor, epidermal growth factor receptor and mitogen-activated protein kinase 8 in diabetic ulcer, and are mainly concentrated in AGE-RAGE, TNF and other signal pathways. Conclusion: Radix Astragali-Lithospermum Erythrorhizon may play the role of inhibiting inflammation, anti-apoptosis, promoting cell proliferation, angiogenesis and immune regulation through multi-components and multitargets, and play a role in the treatment of diabetic ulcer. [ABSTRACT FROM AUTHOR]

Objective: Based on data mining, to explore the clinical characteristics of traditional Chinese medicine ointment in the treatment of diabetic foot ulcer. Methods: to search the literatures about the treatment of diabetic foot ulcer by traditional Chinese medicine ointment in CNKI, Wangfang and VIP. In this paper, the prescription of traditional Chinese medicine ointment in the literature of treating diabetic foot ulcer was extracted, and the database of prescription was established by using v2.5 to analyze and mine the rule of medication. Results: 95 prescriptions for diabetic foot ulcer were sorted out, involving 158 prescriptions. Among them, dragon's blood and Lithospermum are the most frequently used drug combinations. The core combination of 10 groups and 5 new prescriptions are obtained by evolution. Conclusion: the treatment of diabetic foot ulcer with traditional Chinese medicine ointment mainly includes clearing away heat, promoting blood circulation and removing blood stasis, and tonifying deficiency. [ABSTRACT FROM AUTHOR]

Gromwell ( Lithospermum erythrorhizon , LE) can mitigate obesity-induced skeletal muscle atrophy in C2C12 myotubes and high-fat diet (HFD)-induced obese mice. The purpose of this study was to investigate the anti-skeletal muscle atrophy effects of LE and the underlying molecular mechanism. C2C12 myotubes were pretreated with LE or shikonin, and active component of LE, for 24 h and then treated with 500 μM palmitic acid (PA) for an additional 24 h. Additionally, mice were fed a HFD for 8 weeks to induced obesity, and then fed either the same diet or a version containing 0.25% LE for 10 weeks. LE attenuated PA-induced myotubes atrophy in differentiated C2C12 myotubes. The supplementation of LE to obese mice significantly increased skeletal muscle weight, lean body mass, muscle strength, and exercise performance compared with those in the HFD group. LE supplementation not only suppressed obesity-induced skeletal muscle lipid accumulation, but also downregulated TNF-α and atrophic genes. LE increased protein synthesis in the skeletal muscle via the mTOR pathway. We observed LE induced increase of mitochondrial biogenesis and upregulation of oxidative phosphorylation related genes in the skeletal muscles. Furthermore, LE increased the expression of peroxisome proliferator-activated receptor-gamma coactivator-1 alpha and the phosphorylation of adenosine monophosphate-activated protein kinase. Collectively, LE may be useful in ameliorating the detrimental effects of obesity-induced skeletal muscle atrophy through the increase of protein synthesis and mitochondrial biogenesis of skeletal muscle.

In recent years, the utilization of smart colorimetric packaging films for monitoring food freshness has garnered significant concentration. However, their limited tensile strength, hydrophobicity, antioxidant, and antibacterial properties have been substantial barriers to widespread adoption. In this study, we harnessed the potential of biodegradable materials, specifically chitosan/polyvinyl alcohol, alongside shikonin extracted from Radix Lithospermi and ZnO nanoparticles, to create a novel colorimetric sensing film. This film boasts an impressive tensile strength of 82.36 ± 2.13 MPa, enhanced hydrophobic characteristics (exemplified by a final contact angle of 99.81°), and outstanding antioxidant and antibacterial properties. It is designed for real-time monitoring of shrimp freshness. Additionally, we verified the effectiveness of this sensing film in detecting shrimp freshness across varying temperature conditions, namely 25 °C and 4 °C was validated through the measurement of total volatile basic nitrogen (TVB-N). Visual inspection unequivocally revealed a transition in color from dark red to purple-light blue and finally to dark bluish providing a clear indication of shrimp spoilage, which demonstrated a strong correlation with the TVB-N content in shrimp measured through standard laboratory procedures. The colorimetric sensing film developed in this study holds great promise for creating smart labels with exceptional antioxidant and antibacterial properties, tailored for visual freshness monitoring of shrimp., Competing Interests: Declaration of competing interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work; there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Shikonin is extracted from the roots of Lithospermum erythrorhizon , and shikonin extracts have been shown to have inhibitory effects on several bacteria. However, shikonin extracts are difficult to formulate because of their poor water solubility. In the present study, we prepared a shikonin dispersion, which was solubilized by the inclusion of β-1,3-1,6 glucan, and analysed the inhibitory effects of this dispersion on Streptococcus mutans and non-mutans streptococci. The shikonin dispersion showed pronounced anti- S. mutans activity, and inhibited growth of and biofilm formation by this bacterium. The shikonin dispersion also showed antimicrobial and antiproliferative effects against non-mutans streptococci. In addition, a clinical trial was conducted in which 20 subjects were asked to brush their teeth for 1 week using either shikonin dispersion-containing or non-containing toothpaste, respectively. The shikonin-containing toothpaste decreased the number of S. mutans in the oral cavity, while no such effect was observed after the use of the shikonin-free toothpaste. These results suggest that shikonin dispersion has an inhibitory effect on S. mutans and non-mutans streptococci, and toothpaste containing shikonin dispersion may be effective in preventing dental caries.

Lithospermum erythrorhizon is a medicinal plant that produces shikonin, a red lipophilic naphthoquinone derivative that accumulates exclusively in roots. The biosynthetic steps required to complete the naphthalene ring of shikonin and its mechanism of secretion remain unclear. Multiple omics studies identified several candidate genes involved in shikonin production. The functions of these genes can be evaluated using virus-induced gene silencing (VIGS) systems, which have been shown advantageous in introducing iRNA genes into non-model plants. This study describes the development of a VIGS system using an apple latent spherical virus (ALSV) vector and a target gene, phytoene desaturase (LePDS1). Virus particles packaged in Nicotiana benthamiana were inoculated into L. erythrorhizon seedlings, yielding new leaves with albino phenotype but without disease symptoms. The levels of LePDS1 mRNAs were significantly lower in the albino plants than in mock control or escape plants. Virus-derived mRNA was detected in infected plants but not in escape and mock plants. Quantitative PCR and deep sequencing analysis indicated that transcription of another hypothetical PDS gene (LePDS2) also decreased in the defective leaves. Virus infection, however, had no effect on shikonin production. These results suggest that virus-based genetic transformation and the VIGS system silence target genes in soil-grown L. erythrorhizon. [ABSTRACT FROM AUTHOR]

Lithospermum erythrorhizon (red gromwell; zicao) is a medicinal and economically valuable plant belonging to the Boraginaceae family. Roots from L. erythrorhizon have been used for centuries based on the antiviral and wound-healing properties produced from the bioactive compound shikonin and its derivatives. More recently, shikonin, its enantiomer alkannin, and several other shikonin/alkannin derivatives have collectively emerged as valuable natural colorants and as novel drug scaffolds. Despite several transcriptomes and proteomes having been generated from L. erythrorhizon, a reference genome is still unavailable. This has limited investigations into elucidating the shikonin/alkannin pathway and understanding its evolutionary and ecological significance. In this study, we obtained a de novo genome assembly for L. erythrorhizon using a combination of Oxford Nanopore long-read and Illumina short-read sequencing technologies. The resulting genome is ∼367.41 Mb long, with a contig N50 size of 314.31 kb and 27,720 predicted protein-coding genes. Using the L. erythrorhizon genome, we identified several additional p-hydroxybenzoate:geranyltransferase (PGT) homologs and provide insight into their evolutionary history. Phylogenetic analysis of prenyltransferases suggests that PGTs originated in a common ancestor of modern shikonin/alkannin-producing Boraginaceous species, likely from a retrotransposition-derived duplication event of an ancestral prenyltransferase gene. Furthermore, knocking down expression of LePGT1 in L. erythrorhizon hairy root lines revealed that LePGT1 is predominantly responsible for shikonin production early in culture establishment. Taken together, the reference genome reported in this study and the provided analysis on the evolutionary origin of shikonin/alkannin biosynthesis will guide elucidation of the remainder of the pathway. [ABSTRACT FROM AUTHOR]

Background: Uveitis is an inflammatory eye condition that threatens vision, and effective anti-inflammatory treatments with minimal side effects are necessary to treat uveitis., Purpose: This study aimed to investigate the effects of Lithospermum erythrorhizon Siebold & Zucc. against endotoxin-induced uveitis in rat and mouse models., Methods: Endotoxin-induced uveitis models of rats and mice were used to evaluate the effects of l. erythrorhizon treatment. Clinical inflammation scores and retinal thickness were assessed in the extract of l. erythrorhizon-treated rats. Histopathological examination revealed inflammatory cell infiltration into the ciliary body. Protein concentration, cellular infiltration, and prostaglandin-E2 levels were measured in the aqueous humor of the extract of l. erythrorhizon-treated rats. Protective effects of l. erythrorhizon on the anterior segment of the eye were examined in mice with endotoxin-induced uveitis. Additionally, we investigated the effect of l. erythrorhizon on the expression of pro-inflammatory cytokines [tumor necrosis factor alpha, interleukin-6, and interleukin-8] in lipopolysaccharide-stimulated THP1 human macrophages and examined the involvement of nuclear factor kappaB/activator protein 1 and interferon regulatory factor signaling pathways. Furthermore, three components of l. erythrorhizon were identified and assessed for their inhibitory effects on LPS-induced inflammation in RAW264.7 macrophage cells., Results: Treatment of the extract of l. erythrorhizon significantly reduced clinical inflammation scores and retinal thickening in rats with endotoxin-induced uveitis. Histopathological examination revealed decreased inflammatory cell infiltration into the ciliary body. The extract of l. erythrorhizon effectively reduced the protein concentration, cellular infiltration, and PG-E2 levels in the aqueous humor of rats with endotoxin-induced uveitis. In mice with endotoxin-induced uveitis, the extract of l. erythrorhizon demonstrated a protective effect on the anterior segment of the eye by reducing inflammation and retinal thickening. The extract of l. erythrorhizon suppressed the expression of pro-inflammatory cytokines (tumor necrosis factor alpha, interleukin-6, and interleukin-8) in lipopolysaccharide-induced inflammation in THP1 human macrophages, by modulating nuclear factor kappaB/activator protein 1 and interferon regulatory factor signaling pathways. Moreover, shikonin, acetylshikonin, and β, β-dimethylacryloylshikonin showed dose-dependent inhibition of nitric oxide, tumor necrosis factor alpha and interleukin-6 production in RAW264.7 macrophage cells., Conclusion: The extract of l. erythrorhizon is a potential therapeutic agent for uveitis management. Administration of the extract of l. erythrorhizon led to reduced inflammation, retinal thickening, and inflammatory cell infiltration in rat and mouse models of uveitis. The compounds (shikonin, acetylshikonin, and β, β-dimethylacryloylshikonin) identified in this study played crucial roles in mediating the anti-inflammatory effects of l. erythrorhizon. These findings indicate that the extract of l. erythrorhizon and its constituent compounds are promising candidates for further research and development of novel treatment modalities for uveitis., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest., (Copyright © 2023. Published by Elsevier GmbH.)

The common gromwell Lithospermum officinale L. is a valuable medicinal plant that has been used in traditional medicine since ancient times. A method to quantify flavonoids in L. officinale leaves by differential spectrophotometry was developed taking advantage of the flavonoid reaction with aluminum chloride. The optimum duration of the reaction was determined, as well as the optimum volume-to-volume ratio between an aqueous ethanolic extract of L. officinale leaves and 2% aluminum chloride (aqueous ethanolic solution). Rutin was used as a standard. The method was validated in terms of specificity, linearity, precision, and accuracy and proved suitable for analytical purposes. The flavonoid content expressed in terms of rutin was found to exceed 2% of the absolutely dry weight in L. officinale leaves over different years of cultivation., (© 2023. Pleiades Publishing, Ltd.)

Plant specialized metabolism emerged from the land colonization by ancient plants, becoming diversified along with plant evolution. To date, more than 1 million metabolites have been predicted to exist in the plant kingdom, and their metabolic processes have been revealed on the molecular level. Previous studies have reported that rates of evolution are greater for genes involved in plant specialized metabolism than in primary metabolism. This perspective introduces topics on the enigmatic molecular evolution of some plant specialized metabolic processes. Two transferase families, BAHD acyltransferases and aromatic prenyltransferases, which are involved in the biosynthesis of paclitaxel and meroterpenes, respectively, have shown apparent expansion. The latter family has been shown to beinvolved in the biosynthesis of a variety of aromatic substances, including prenylated coumarins in citrus plants and shikonin in Lithospermum erythrorhizon. These genes have evolved in the development of each special subfamily within the plant lineage. The broadness of substrate specificity and the exon-intron structure of their genes may provide hints to explain the evolutionary process underlying chemodiversity in plants. [ABSTRACT FROM AUTHOR]

One new furylhydroquinone derivative (1) and seven known compounds (2–8) were isolated from the roots of Lithospermum erythrorhizon Sieb. et Zucc (Boraginaceae). The structure of 1 was elucidated by extensive spectroscopic methods using NMR and MS. The absolute configuration of shikonofuran J (1) was unambiguously determined by aid of comparison experimental ECD with predicted ECD spectra. All the isolates were tested for their inhibitory activities against IL-6 production in HaCaT cells stimulated by tumor necrosis factor (TNF)-α. It was found that gracicleistanthoside (5) and uridine (7) remarkably down-regulated the TNF-α-induced synthesis of interleukin-6 (IL-6), a pro-inflammatory cytokine associated with cutaneous inflammation, in HaCaT cells. [ABSTRACT FROM AUTHOR]

A transplanted organ is usually rejected without any major immunosuppressive treatment because of vigorous alloimmune responsiveness. However, continuous global immunosuppression may cause severe side effects, including nephrotoxicity, tumors, and infections. Therefore, it is necessary to seek novel immunosuppressive agents, especially natural ingredients that may provide sufficient efficacy in immunosuppression with minimal side effects. Shikonin is a bioactive naphthoquinone pigment, an ingredient originally extracted from the root of Lithospermum erythrorhizon. Previous studies have shown that shikonin regulates immunity and exerts anti-inflammatory effects. In particular, it can ameliorate arthritis in animal models. However, it is unclear whether shikonin inhibits alloimmunity or allograft rejection. In this study and for the first time, we demonstrated that shikonin significantly prolonged the survival of skin allografts in wild-type mice. Shikonin increased the frequencies of CD4+Foxp3+ regulatory T cells (Tregs) post-transplantation and induced CD4+Foxp3+ Tregs in vitro as well. Importantly, depleting the Tregs abrogated the extension of skin allograft survival induced by shikonin. It also decreased the frequencies of CD8+CD44highCD62Llow effector T cells and CD11c+CD80+/CD11c+CD86+ mature DCs after transplantation. Moreover, we found that shikonin inhibited the proliferation of T cells in vitro and suppressed their mTOR signaling. It also reduced the gene expression of pro-inflammatory cytokines, including IFNγ, IL-6, TNFα, and IL-17A, while increasing the gene expression of anti-inflammatory mediators IL-10, TGF-β1, and indoleamine-2, 3-dioxygenase (IDO) in skin allografts. Further, shikonin downregulated IDO protein expression in skin allografts and DCs in vitro. Taken together, shikonin inhibits allograft rejection via upregulating CD4+Foxp3+ Tregs. Thus, shikonin is a novel immunosuppressant that could be potentially used in clinical transplantation. [ABSTRACT FROM AUTHOR]

Plants produce a large variety of specialized (secondary) metabolites having a wide range of hydrophobicity. Shikonin, a red naphthoquinone pigment, is a highly hydrophobic metabolite produced in the roots of Lithospermum erythrorhizon, a medicinal plant in the family Boraginaceae. The shikonin molecule is formed by the coupling of p -hydroxybenzoic acid and geranyl diphosphate, catalyzed by a membrane-bound geranyltransferase LePGT at the endoplasmic reticulum, followed by cyclization of the geranyl chain and oxidations; the latter half of this biosynthetic pathway, however, has not yet been clarified. To shed light on these steps, a proteome analysis was conducted. Shikonin production in vitro was specifically regulated by illumination and by the difference in media used to culture cells and hairy roots. In intact plants, however, shikonin is produced exclusively in the root bark of L. erythrorhizon. These features were utilized for comparative transcriptome and proteome analyses. As the genome sequence is not known for this medicinal plant, sequences from de novo RNA-seq data with 95,861 contigs were used as reference for proteome analysis. Because shikonin biosynthesis requires copper ions and is sensitive to blue light, this methodology identified strong candidates for enzymes involved in shikonin biosynthesis, such as polyphenol oxidase, cannabidiolic acid synthase-like and neomenthol dehydrogenase-like proteins. Because acetylshikonin is the main end product of shikonin derivatives, an O -acetyltransferase was also identified. This enzyme may be responsible for end product formation in these plant species. Taken together, these findings suggest a putative pathway for shikonin biosynthesis. [ABSTRACT FROM AUTHOR]

The imbalance between the proliferation and apoptosis of pulmonary artery smooth muscle cells (PASMCs) is of importance in pulmonary vascular remodeling. Shikonin, a naphthoquinone compound extracted from the Chinese medicinal herb Lithospermum erythrorhizon, inhibits the proliferation of rat smooth muscle cells (SMCs). The present study was designed to investigate the effects of shikonin on the proliferation of rat PASMCs and the possible mechanisms involved. Rat PASMCs were cultured under the following five treatment conditions: Normal control; hypoxia for 24 h; hypoxia + 1 µM shikonin for 24 h; hypoxia + 2 µM shikonin for 24 h; and hypoxia + 4 µM shikonin for 24 h. The viability of PASMCs was measured using the Cell Counting Kit-8 assay, the mRNA expression of nestin (NES) in each group was measured by reverse transcription-polymerase chain reaction and the protein expression of NES was measured by western blotting. The proliferation of hypoxic PASMCs transfected with NES-specific small interfering (si)RNA decreased compared with the non-transfected group. These results indicated that hypoxia induced the proliferation of PASMCs through the enhancement of NES expression. The treatment of hypoxic PASMCs with shikonin resulted in a significant downregulation of NES expression and the inhibition of PASMC proliferation. [ABSTRACT FROM AUTHOR]

Background:DF formula is a herbal preparation comprised three medicinal herbs, namely, Ephedra intermedia, Rheum palmatum, and Lithospermum erythrorhizon, which is being used for the treatment of obesity and liver fibrosis in Korean local clinics. Objective: Since the abovementioned three herbs exist with different proportions in DF formula and their chemical markers have different physiochemical properties; it is quite challenging to develop an analytical methodology for the determination of these chemical markers. Materials and Methods: For the analysis of the three herbs, five chemicals, (+)-pseudoephedrine (1) and (−)-ephedrine (2) for E. intermedia, aloe-emodin (3), and chrysophanol (4) for R. palmatum, and shikonin (5) for L. erythrorhizon, were selected for method validation of DF formula, and the analytical conditions were optimized and validated using high-performance liquid chromatography coupled with an ultraviolet detector (HPLC-UV). Results: The specificities for the five compounds 1–5 were determined by their UV absorption spectra (1–4: 215 nm and 5: 520 nm). Their calibration curves showed good linear regressions with high correlation coefficient values (R2 > 0.9997). The limits of detection of these five markers were in the range 0.4–2.1 ng/mL, with the exception of 5 (12.7 ng/mL). The intraday variability for all the chemical markers was less than a Relative standard deviation (RSD) of 3%, except for 5 (RSD = 12.6%). In the case of interday analysis, 1 (1.0%), 2 (3.1%), and 4 (3.7%) showed much lower variabilities (RSD < 5%) than 3 (7.6%) and 5 (8.2%). Moreover, the five chemical markers showed good recoveries with good accuracies in the range of 90%–110%. Conclusions: The developed HPLC-UV method for the determination of the five chemical markers of the components of DF formula was validated. Abbreviations used: EP: (−)-ephedrine; PSEP: (+)-pseudoephedrine; HPLC: High-performance liquid chromatography; UV: Ultraviolet; LOD: Limit of detection; LOQ: Limit of quantification; RSD: Relative standard deviation. [ABSTRACT FROM AUTHOR]

Abstract Lithospermum L. (Boraginaceae) includes approximately 60 species of which 43 occur in Mexico, with 32 endemic to the country. Through morphological and molecular investigations, the species of the genus are taxonomically revised. A broad generic circumscription is recognized and includes species traditionally recognized as members of Lithospermum as well as other New World Lithospermeae. Three new species are described from northern Mexico—Lithospermum chihuahuanum J. I. Cohen, Lithospermum kelloggianum J. I. Cohen, and Lithospermum tenerum J. I. Cohen—and each is known from only its type specimen. Lasiarrhenum confundum B. L. Turner is transferred to Lithospermum confundum (B. L. Turner) J. I. Cohen on the basis of flowers similar to those of Lithospermum trinervium (Lehm.) J. I. Cohen. The type for Pentalophus A. DC. is designated. Lectotypes are designated for Lithospermum albicans Greene [= Lithospermum incisum Lehm.], Lithospermum approximatum Brand [= Lithospermum distichum Ortega], Lithospermum breviflorum Engelm. & A. Gray [= Lithospermum incisum Lehm.], Lithospermum cobrense Greene, Lithospermum cognatum Greene ex Spengle [= Lithospermum multiflorum Torr. ex A. Gray], Lithospermum euryphyllum Brand [= Lithospermum oblongifolium Greenm.], Lithospermum galeottii Brand [= Lithospermum calycosum (J. F. Macbr.) I. M. Johnst.], Lithospermum gentianoides Brand [= Lithospermum obovatum J. F. Macbr.], Lithospermum hoyasense Brand [= Lithospermum calcicola B. L. Rob.], Lithospermum laevigatum Sessé & Moc. [= Lithospermum distichum Ortega], Lithospermum madrense Brand [= Lithospermum calcicola B. L. Rob.], Lithospermum matamorense DC., Lithospermum nelsonii Greenm., Lithospermum obtusiflorum Sessé & Moc. [= Lithospermum discolor M. Martens & Galeotti], Lithospermum sordidum Brand [= Lithospermum distichum Ortega], and Lithospermum spathulatum M. Martens & Galeotti [= Lithospermum distichum Ortega]. Lithospermum viride Greene is neotypified. [ABSTRACT FROM AUTHOR]

Abstract: Shikonin and its derivatives are important medicinal secondary metabolites accumulating in roots of Lithospermum erythrorhizon. Although some membrane proteins have been identified as transporters of secondary metabolites, the mechanisms underlying shikonin transport and accumulation in L. erythrorhizon cells still remain largely unknown. In this study, we isolated a cDNA encoding LeMRP, an ATP‐binding cassette transporter from L. erythrorhizon, and further investigated its functions in the transport and biosynthesis of shikonin using the yeast transformation and transgenic hairy root methods, respectively. Real‐time PCR was applied for expression analyses of LeMRP and shikonin biosynthetic enzyme genes. Functional analysis of LeMRP using the heterologous yeast cell expression system showed that LeMRP could be involved in shikonin transport. Transgenic hairy roots of L. erythrorhizon demonstrated that LeMRP overexpressing hairy roots produced more shikonin than the empty vector (EV) control. Real‐time PCR results revealed that the enhanced shikonin biosynthesis in the overexpression lines was mainly caused by highly up‐regulated expression of genes coding key enzymes (LePAL, HMGR, Le4CL and LePGT) involved in shikonin biosynthesis. Conversely, LeMRP RNAi decreased the accumulation of shikonin and effectively down‐regulated expression level of the above genes. Typical inhibitors of ABC proteins, such as azide and buthionine sulphoximine, dramatically inhibited accumulation of shikonin in hairy roots. Our findings provide evidence for the important direct or indirect role of LeMRP in transmembrane transport and biosynthesis of shikonin. [ABSTRACT FROM AUTHOR]

Hyperglycemia-induced oxidative stress is thought to play a critical role in the pathogenesis of diabetic nephropathy (DN). Treating high-glucose (HG)-induced proximal tubule injury has become a patential therapeutic option to attenuate the onset and progression of DN. The present study aimed to investigate the renoprotective effect of shikonin, the chief active compound extracted from the roots of the traditional Chinese herb Lithospermum erythrorhizon, on HG-induced cytotoxicity in NRK-52E cells. Treating cells with HG significantly reduce cell viability while also significantly increasing content of reactive oxygen species (ROS). Treating the cells with shikonin improved these changes induced by HG. Shikonin strongly stabilized mitochondrial membrane potential in HG-induced NRK-52E cells. In addition, treatment with shikonin upregulated antioxidant system in response to ROS by increasing levels of SOD and CAT. Furthermore, shikonin also strongly decreased the levels of activated caspase-3, Bax and p-GSK-3β while increased the p-AKT level. These findings provide that the renoprotective effects of shikonin against HG-induced cytotoxicity in NRK-52E cells may be mediated in inhibiting oxidative stress through activating of the AKT signalling pathway. [ABSTRACT FROM AUTHOR]

Objective: To examine specific molecular mechanisms involved in modulating hepatic lipogenesis and mitochondria biogenesis signals by Lithospermum erythrorhizon (gromwell) root extract.Methods: Stable cell lines with luciferase reporter constructs were generated to examine sterol regulatory element binding protein 1c (SREBP1c) and peroxisome proliferator-activated receptor gamma, coactivator 1 (PGC1) α promoter activity and estrogen-related receptor (ERR) α response element activity. Gene expression of SREBP1c, stearoyl coenzyme A desaturase 1, and PGC1α was measured by using reverse transcription polymerase chain reaction. Lipogenesis was measured in human hepatoma cells with Nile red staining and flow cytometry. Phosphorylation of AMP-activated protein kinase (AMPK) α was determined by using ELISA and Western blot.Results: Gromwell root extract and its naphthoquinones dose-dependently repressed high glucose and liver X receptor α induction of SREBP1c promoter activity and gene expression. Hepatic lipogenesis was repressed, and PGC1α promoter and gene expression and ERRα response element activity were increased by gromwell root extract. Gromwell root extract, shikonin, and α-methyl-n-butyrylshikonin increased AMPKα phosphorylation, and inhibition of AMPK blunted the repression in SREBP1c promoter activity by gromwell root extract and its naphthoquinones.Conclusions: Data suggest that gromwell root extract and its naphthoquinones repress lipogenesis by increasing the phosphorylated state of AMPKα and stimulating mitochondrial biogenesis signals. [ABSTRACT FROM AUTHOR]

Background: Shikonin is a naphthoquinone secondary metabolite with important medicinal value and is found in Lithospermum erythrorhizon. Considering the limited knowledge on the membrane transport mechanism of shikonin, this study investigated such molecular mechanism. Results: We successfully isolated an ATP-binding cassette protein gene, LeMDR, from L. erythrorhizon. LeMDR is predominantly expressed in L. erythrorhizon roots, where shikonin accumulated. Functional analysis of LeMDR by using the yeast cell expression system revealed that LeMDR is possibly involved in the shikonin efflux transport. The accumulation of shikonin is lower in yeast cells transformed with LeMDR-overexpressing vector than that with empty vector. The transgenic hairy roots of L. erythrorhizon overexpressing LeMDR (MDRO) significantly enhanced shikonin production, whereas the RNA interference of LeMDR (MDRi) displayed a reverse trend. Moreover, the mRNA expression level of LeMDR was up-regulated by treatment with shikonin and shikonin-positive regulators, methyl jasmonate and indole-3-acetic acid. There might be a relationship of mutual regulation between the expression level of LeMDR and shikonin biosynthesis. Conclusions: Our findings demonstrated the important role of LeMDR in transmembrane transport and biosynthesis of shikonin. [ABSTRACT FROM AUTHOR]

Shikonin is a naphthoquinone pigment extracted from roots of Lithospermum erythrorhizon Sieb. et Zucc. (Boraginaceae), and possesses various pharmaceutical activities, such as anti-inflammation and anti-cancer effects. In addition, shikonin as a natural red colorant for food garnishment and cosmetics ingredient is widely used in the world. However, the inhibition risk of shikonin on cytochrome P450 (CYP) remains unclear. The aim of this study was to investigate the potential inhibition of shikonin against CYP1A2, CYP2B1/6, CYP2C9/11, CYP2D1/6, CYP2E1 and CYP3A2/4 activities in human and rat liver microsomes through cocktail approach in vitro. The results demonstrated that shikonin exhibited no time-dependent inhibition of CYP activities. In human liver microsomes, shikonin was not only a mixed inhibitor of CYP1A2, CYP2B6, CYP2C9, CYP2D6 and CYP3A4, but also a competitive inhibitor of CYP2E1, with K i values no more than 7.72 μM. In rat liver microsomes, shikonin also exhibited the mixed inhibition on CYP1A2, CYP2B1, CYP2C11, CYP2D1, and the competitive inhibition on CYP2E1. Interestingly, shikonin presented an atypical kinetic inhibition of CYP3A2-mediated midazolam 1-hydroxylation in rats. In conclusion, the relatively low K i values of shikonin would have a high risk potential to cause the possible toxicity, especially drug-drug or food-drug interactions based on the potent inhibition of CYP enzymes. [ABSTRACT FROM AUTHOR]

This study proposes an optimization process of the Lithospermumerythrorhizon (LE) extraction with a higher purity of shikonin (SK). The influence of extraction temperature on the concentration of SK is examined, and an in vitro cell viability assay is used to examine the optimal concentration of SK. Afterwards, polyvinyl alcohol (PVA)/LE solutions at ratios of 90/10, 80/20, and 70/30 w/w are electrospun into LE electrospun nanofibrous membranes (LENMs). The optimal manufacture parameters of LENMs are evaluated based on the test results of in vitro drug release test and cell viability assay. The optimal concentration occurs when the extraction temperature is -10°C. The purity of the LE extract reaches 53.8% and the concentration of SK is 1.07 mg/mL. Moreover, the cell viability of nanofibrous membranes significantly increases to 136.8% when 0.7 μM SK is used. The diameter of nanofibers of LENM is decreased by 43.9% when the ratio of PVA solution to LE extract is 70/30 (w/w). 80/20 (w/w) LENM has the maximum amount of drug release of 79% for a continuous period of 48 h. In particular, 90/10 (w/w) LENM can create the maximum cell proliferation of 157.5% in a 24-h in vitro cell viability assay. This suggests that LENM has great potential to be used in facilitating tissue regeneration and wound healing. [ABSTRACT FROM AUTHOR]

Hypoxia enhances the development of solid tumors. Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor that is dominantly expressed under hypoxia in solid tumor cells and is a key factor of tumor regulation. HIF-1α regulates several target genes involved in many aspects of cancer progression, including angiogenesis, metastasis, and cell proliferation, as well as imparting resistance to cancer treatment. In this study, we assessed shikonin, which derives from the traditional medical herb Lithospermum erythrorhizon , for its anti-cancer effects in hypoxia-induced human colon cancer cell lines. Shikonin showed potent inhibitory activity against hypoxia-induced HIF-1α activation in various human cancer cell lines and efficient scavenging activity of hypoxia-induced reactive oxygen species in tumor cells. Further analysis revealed that shikonin inhibited HIF-1α protein synthesis without affecting the expression of HIF-1α mRNA or degrading HIF-1α protein. It was subsequently shown to attenuate the activation of downstream mTOR/p70S6K/4E-BP1/eIF4E kinase. Shikonin also dose-dependently caused the cell cycle arrest of activated HCT116 cells and inhibited the proliferation of HCT116 and SW620 cells. Moreover, it significantly inhibited tumor growth in a xenograft modal. These findings suggest that shikonin could be considered for use as a potential drug in human colon cancer therapy. [ABSTRACT FROM AUTHOR]

A novel hollow-fiber liquid-phase microextraction based on oil-in-salt was proposed and introduced for the simultaneous extraction and enrichment of the main active compounds of hesperidin, honokiol, shikonin, magnolol, emodin, and β,β′-dimethylacrylshikonin in a formula of Zi-Cao-Cheng-Qi decoction and the single herb, Fructus Aurantii Immaturus, Cortex Magnoliae Officinalis, Radix et Rhizoma, and Lithospermum erythrorhizon, composing the formula prior to their analysis by high-performance liquid chromatography. The results obtained by the proposed procedure were compared with those obtained by conventional hollow-fiber liquid-phase microextraction, and the proposed procedure mechanism was described. In the procedure, a hollow-fiber segment was first immersed in organic solvent to fill the solvent in the fiber lumen and wall pore, and then the fiber was again immersed into sodium chloride solution to cover a thin salt membrane on the fiber wall pore filling organic solvent. Under the optimum conditions, the enrichment factors of the analytes were 0.6-109.4, linearities were 0.002-12 μg/mL with r2 ≥ 0.9950, detection limits were 0.6-12 ng/mL, respectively. The results showed that oil-in-salt hollow-fiber liquid-phase microextraction is a simple and effective sample pretreatment procedure and suitable for the simultaneous extraction and concentration of trace-level active compounds in traditional Chinese medicine. [ABSTRACT FROM AUTHOR]

Background: Shikonin, a natural naphthoquinone pigment purified from Lithospermum erythrorhizon, induces necroptosis in various cancer types, but the mechanisms underlying the anticancer activity of shikonin in lung cancer are not fully understood. This study was designed to clarify whether shikonin causes necroptosis in non-small cell lung cancer (NSCLC) cells and to investigate the mechanism of action.Methods: Multiplex and caspase 8 assays were used to analyze effect of shikonin on A549 cells. Cytometry with annexin V/PI staining and MTT assays were used to analyze the mode of cell death. Western blotting was used to determine the effect of shikonin-induced necroptosis and autophagy. Xenograft and orthotopic models with A549 cells were used to evaluate the anti-tumor effect of shikonin in vivo.Results: Most of the cell death induced by shikonin could be rescued by the specific necroptosis inhibitor necrostatin-1, but not by the general caspase inhibitor Z-VAD-FMK. Tumor growth was significantly lower in animals treated with shikonin than in the control group. Shikonin also increased RIP1 protein expression in tumor tissues. Autophagy inhibitors, including methyladenine (3-MA), ATG5 siRNA, and bafilomycin A, enhanced shikonin-induced necroptosis, whereas RIP1 siRNA had no effect on the apoptotic potential of shikonin.Conclusions: Our data indicated that shikonin treatment induced necroptosis and autophagy in NSCLC cells. In addition, the inhibition of shikonin-induced autophagy enhanced necroptosis, suggesting that shikonin could be a novel therapeutic strategy against NSCLC. [ABSTRACT FROM AUTHOR]

Genes controlling the morphological, micromorphological, and physiological components of the breeding system distyly have been hypothesized, but many of the genes have not been investigated throughout development of the two floral morphs. To this end, the present study is an examination of comparative transcriptomes from three stages of development for the floral organs of the morphs of Lithospermum multiflorum. Transcriptomes of flowers of the two morphs, from various stages of development, were sequenced using an Illumina HiSeq 2000. The floral transcriptome of L. multiflorum was assembled, and differential gene expression (DE) was identified between morphs, throughout development. Additionally, Gene Ontology (GO) terms for DE genes were determined. Fewer genes were DE early in development compared to later in development, with more genes highly expressed in the gynoecium of the SS morph and the corolla and androecium of the LS morph. A reciprocal pattern was observed later in development, and many more genes were DE during this latter stage. During early development, DE genes appear to be involved in growth and floral development, and during later development, DE genes seem to affect physiological functions. Interestingly, many genes involved in response to stress were identified as DE between morphs. [ABSTRACT FROM AUTHOR]

β,β-dimethylacryloyl alkannin (DMA) is a key component of Lithospermum and possesses good efficacy for treating psoriasis. DMA inhibits activated dendritic cells (DCs), but the mechanism is unknown. Therefore, this study aimed to explore the modulation of the TLR7/8 pathway by DMA in psoriasis-activated DCs. Models of psoriasis-like skin lesions were established using BALB/c mice; 8 mice were treated with DMA (2.5 mg/kg). Bone marrow cells were isolated and induced into DCs using R848, a TLR7/8 agonist. Splenic CD11c + cells were detected by flow cytometry. Skin CD11c + cells were detected by immunofluorescence. TLR7, TLR8, MYD88, and IRAKM proteins were detected by Western blot. The effects of DMA on surface molecules of DCs were observed by flow cytometry. mRNA expression of inflammatory factors was detected by qRT-PCR. Secreted cytokines were detected by cytometric bead array. Compared with the model group, psoriasis-like skin lesions were alleviated by DMA, the splenic CD11c + cells were significantly decreased ( P < 0.01), and CD11c + cell numbers in skin lesions were decreased ( P < 0.01). Expression levels of TLR7, MYD88, and IRAKM were significantly decreased ( P < 0.05). R848-stimulated DCs showed increased expression of I-A/I-E, CD80, and CD86 ( P < 0.01), increased IL-23 and IL-1β mRNA and secretion ( P < 0.05), and increased TLR7, TLR8, MYD88, and IRAKM expression ( P < 0.01); DMA inhibited all of these effects of the TLR7/8 pathway activation by R848 ( P < 0.05). In conclusion, DMA could inhibit psoriasis-activated DCs via the TLR7/8 pathway. [ABSTRACT FROM AUTHOR]

A novel quantitative 1H NMR (Q-NMR) combined with HPLC-MS method has been proposed for investigating the degradation process of traditional Chinese medicine (TCM) components. Through this method, in-situ monitoring of dynamics degradation process of lithospermic acid (LA), one of the popular polyphenolic acids in TCM, was realized under low oxygen condition. Additionally, this methodology was proved to be simple, rapid and specific. Degradation kinetic runs have been carried out to systematically investigate the effects of two key environmental factors, initial pH values and temperatures. Eight main degradation products of LA were detected, seven of which were tentatively structural elucidated with the help of both NMR and LC-MS in this work and salvianolic acid A (Sal A) was the primary degradation product of LA. A possible degradation pathway of LA was proposed, subsequently. The results showed that the degradation of LA followed pseudo-first-order kinetics. The apparent degradation kinetic constants increased as the initial pH value of the phosphate buffer increased. Under the given conditions, the rate constants of overall degradation as a function of temperature obeyed the Arrhenius equation. Our results proved that the Q-NMR combined with HPLC-MS method can be one of the most promising techniques for investigating degradation process of active components in TCM. [ABSTRACT FROM AUTHOR]

Objectives Recently, shikonin derivatives from Lithospermum erythrorhizon have been suggested as potential chemotherapeutic agents against numerous types of cancers in addition to their traditional uses, e.g., as anti-inflammatory agents. Acetylshikonin, one of shikonin derivatives, has also been reported to possess anticancer activity. However, few studies of the effectiveness of acetylshikonin against cancer cells have been conducted, and there are no studies of oral cancers. In this study, we investigated the usefulness of acetylshikonin as a treatment regimen for oral cancers by observing the growth inhibitory function of acetylshikonin and the involved mechanisms. Designs The viability, cell cycle, and ratio of apoptotic cells of oral squamous cell carcinoma (OSCC) cells were observed after treatment with acetylshikonin using MTT assay, flow cytometric analysis, and Annexin V/PI staining, respectively. In addition, molecular changes of apoptosis-related pathways and the role of reactive oxygen species (ROS) were analyzed in acetylshikonin-treated cells. Results We observed that acetylshikonin significantly suppressed the growth of OSCC cells by inducing apoptotic cell death, and acetylshikonin affected the viability of a normal keratinocyte cell line HaCaT to a lesser degree, suggesting that acetylshikonin may be a good chemotherapeutic reagent with less toxicity to normal tissues. In addition, we found that acetylshikonin-induced apoptosis of OSCC cells is mediated by ROS as well as G2 cell cycle arrest. ROS production in response to acetylshikonin treatment enhanced the phosphorylation of JNK and p38 MAPK, which are in the major pathways of apoptotic cell death mechanisms. Conclusions In summary, our data suggest that acetylshikonin is a strong candidate for use as a selective chemotherapeutic agent for the treatment of OSCC. [ABSTRACT FROM AUTHOR]

During the past decades, agro-biodiversity has markedly declined and some species are close to extinction in large parts of Europe. Reintroduction of rare arable plant species in suitable habitats could counteract this negative trend. The study investigates optimal sowing rates of three endangered species ( Legousia speculum-veneris (L.) Chaix, Consolida regalis Gray, and Lithospermum arvense L.), in terms of establishment success, seed production, and crop yield losses., A field experiment with partial additive design was performed in an organically managed winter rye stand with study species added in ten sowing rates of 5-10,000 seeds m−2. They were sown as a single species or as a three-species mixture (pure vs. mixed sowing) and with vs. without removal of spontaneous weeds. Winter rye was sown at a fixed rate of 350 grains m−2. Performance of the study species was assessed as plant establishment and seed production. Crop response was determined as grain yield., Plant numbers and seed production were significantly affected by the sowing rate, but not by sowing type (pure vs. mixed sowing of the three study species), and weed removal. All rare arable plant species established and reproduced at sowing rates >25 seeds m−2, with best performance of L. speculum-veneris. Negative density effects occurred to some extent for plant establishment and more markedly for seed production., The impact of the three study species on crop yield followed sigmoidal functions. Depending on the species, a yield loss of 10% occurred at >100 seeds m−2., Synthesis and applications: The study shows that reintroduction of rare arable plants by seed transfer is a suitable method to establish them on extensively managed fields, for example, in organic farms with low nutrient level and without mechanical weed control. Sowing rates of 100 seeds m−2 for C. regalis and L. arvense, and 50 seeds m−2 for L. speculum-veneris are recommended, to achieve successful establishment with negligible crop yield losses. [ABSTRACT FROM AUTHOR]